A novel approach has been developed to study the reprogramming of protein kinases en masse allowing ~40-60% of the expressed kinome assayed in a single mass spectroscopy run. Our methods utilize Multiplexed Inhibitor Beads (MIBs), consisting of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors. The technique allows interrogation of kinases known by sequence but which have been understudied due to lack of biologic or phenotypic knowledge or the availability of reagents. MIB/MS identified a kinome response signature to a select MEK1/2 inhibitor AZD6244 that is currently in clinical testing for triple negative breast cancer. The only defined substrate for MEK1/2 are ERK1 and 2, yet we observed changes in activity of kinases in every subfamily of the kinome in response to MEK inhibition. Kinome assessment showed a time-dependent reprogramming that involved an early loss of ERK feedback regulation of RAF and MEK that allowed upstream reactivation of the MEK-ERK pathway. The time dependent change in MIB binding of specific receptor tyrosine kinases (RTKs) such as PDGFR and DDR1 was readily detected and provided the critical experimental observation that MEK inhibition was driving the expression and activation of multiple RTKs. c-Myc degradation was a key mechanism mediating kinome reprogramming. The fact that multiple RTKs are activated in response to MEK inhibition demonstrates the difficulty in using single kinase inhibitors to arrest tumor progression. The aims of this proposal include: 1. Define kinome activation state in the human basal/claudin-low like SUM159 cell line and the pre-clinical C3Tag GEMM for basal/claudin-low breast cancer. Kinase signatures will be defined in response to MEK1/2 and PI3K inhibitors alone and in combination, which are currently in clinical testing. 2. Define mechanisms of kinome reprogramming in MEK and PI3K inhibitor resistant tumors and cell lines. 3. Develop rational predictions of kinase inhibitor combinations based on kinome signatures of sensitive and resistant tumors for testing in the C3Tag GEMM for basal/claudin-low breast cancer. The goal is to define combination therapies that overcome resistance and cause apoptosis and tumor regression.